Characterization and Regulation of Catabolic Genes

Critical Reviews in Microbiology, 25(4):245–273 (1999)

Characterization and Regulation of Catabolic Genes Atul K. Johri,1* Meenakshi Dua,2 Ajay Singh,3 N. Sethunathan,4 and Raymond L. Legge 1 1

Department of Chemical Engineering, Faculty of Engineering, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada; 2Molecular Biology Laboratory, Department of Zoology, University of Delhi, Delhi, Delhi-7, India; 3Microbial Biotechnology Laboratory, Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada; 4Division of Microbiology, Indian Agricultural Research Institute, PUSA, New Delhi-67, India *

Corresponding author’s present address: Dr. A. K. Johri, c/o Prof. A. N. Maitra, Department of Chemistry, University of Delhi, Delhi-110007, India; Fax: 91-11-7256541, 91-11-7256593; E-mail: [email protected]

TABLE OF CONTENTS I. Introduction ..................................................................................................................... 246 II. Toluene and Xylene ......................................................................................................... 247 A. Catabolic Genes......................................................................................................... 247 B. Gene Regulation ........................................................................................................ 249 III. Chlorobenzoates ............................................................................................................... 250 A. Organization and Regulation of Genes .................................................................. 251 IV. Chlorophenoxyacetates ................................................................................................... 252 A. Organization and Regulation of Genes .................................................................. 253 V. Polychlorinated Biphenyls .............................................................................................. 255 A. Organization of Catabolic Genes ............................................................................ 255 VI. Naphthalene, Phenanthrene, and Anthracene ............................................................. 258 A. Organization of Genes .............................................................................................. 258 B. Regulatory Genes ...................................................................................................... 258 VII. Parathion .......................................................................................................................... 261 VIII. Hexachlorocyclohexane ................................................................................................... 261 IX. Recent Developments, Conclusions, and Future Prospects ........................................ 262 Acknowledgments ............................................................................................................ 263 References ......................................................................................................................... 264

1040-841X/99/$.50 © 1999 by CRC Press LLC

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ABSTRACT: Although a wide range of microorganisms have been discovered that are able to degrade highly stable, toxic xenobiotics, still many pollutants persist in the environment. Recent advances in the field of r-DNA technology has provided solutions to these problems. One important factor limiting the bioremediation of sites contaminated with certain hazardous wastes is the slow rate of degradation. This slow rate limits the practicality of using bacteria in remediating contaminated sites. It is possible to extend the range of substrates that an organism can utilize. It is even possible to endow an organism with the ability to degrade a predetermined range of xenobiotics. Because biotechnological processes are based on natural activities of microorganisms and costitute variations in classic domestic waste treatment processes, they are publicly more accepted. This is an area where genetic engineering can make a marked improvement by manipulating catabolic genes of microorgnisms. Advances in r-DNA technology have opend up new avenues to move toward the goal of genetically engineered microorganisms to function as “designer biocatalysts” in which certain desirable biodegradation pathways or enzymes from different organisms are brought together in a single host with the aim of performing specific detoxification. In the last 2 decades much progress has been made in this direction, and as a result catabolic genes have been cloned and characterized for organochlorines, polychlorinatedbiphenyls, chlrobenzoates, naphthalene etc. The aim of this review is to provide an insight in the recent advances made on characterization and expression of catabolic genes that encode the degradation /detoxification of these pesistent and toxic xenobiotic compounds. KEY WORDS: catabolic genes, degradation, xenobiotic, enzymes, plasmid.

I. INTRODUCTION The use of xenobiotics continues to be controversial because they are indispensable yet pose a problem of harmful persistence in the environment. This calls for devising methods of remediation using microorganisms known naturally to degrade them. However, a prerequisite for this is understanding the genetic infrastructure of these microbes. Recombinant DNA technology has helped considerably in understanding the molecular biology of catabolic genes. Catabolic genes and their regulation have been investigated for some time. Important xenobiotics like toluene, chlorobenzoates, chlorophenoxyacetates, polychlorinated biphenyls, and hexachlorocyclohexane have been investigated for some time by a number of research groups. The objective of this review is to provide a comprehensive overview of the work to date on the characterization and regulation of catabolic genes responsible for the degradation of some

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well-known xenobiotics. The genetic control for some of these xenobiotics is only partially understood, yet development of GEMs, gene containment systems, and molecular tools like biosensors offer promise for the future. The development of recombinant DNA techniques has brought about a phenomenal revolution in the field of biological sciences. Although the application of r-DNA techniques in identification and characterization of catabolic genes in microbes started in the early 1970s, and studies on the expression and regulation of catabolic genes for the degradation and detoxification of xenobiotics have been partially completed for chlorophenoxyacetates, PCBs, parathion, naphthalene, chlorobenzoates, and benzoates, etc., little has be done in the area of genetic manipulations of catabolic genes responsible for the degradation of organochlorines.1 Following is a recent account of the characterization and regulation of catabolic genes responsible for the degradation of various xenobiotics.

II. TOLUENE AND XYLENE Genetic information on the degradation of these compounds has paved the way for a fundamental understanding of the molecular biology of catabolic genes.

A. Catabolic Genes Five unique bacterial pathways that result in oxygenase-catalyzed hydroxylation of toluene have been described.2 One involves the oxidation of toluene through benzyl alcohol, benzaldehyde, and benzoate to catechol and is the only route known to be encoded by a catabolic plasmid TOL.3 The remaining pathways initiate toluene oxidation through the hydroxylation of aromatic ring carbons via either mono- or dioxygenases. Only one toluene dioxygenase, the toluene 2,3-dioxygenase of Pseudomonas putida F1, has been described. Toluene 2,3-dioxygenase produces cis-toluene-2,3dihydrodiol from toluene with the addition of a single diatomic oxygen.4 Toluene monooxygenases that hydroxylate the aromatic nucleus at all three possible positions, producing ortho-, meta-, and para-cresol, have been described. These include the tolueneortho-monooxygenase of Burkholderia cepacia G4,5 the toluene meta-mono-oxygenase of P. pickettii PK01,6 and the toluene para-monooxygenase of P. mendocina KR1.7 The genes that encode these oxy-genases have also been described.6,8–11 Toluene 2,3dioxygenase has been shown to be a complex of three proteins, a reductase, a ferredoxin reductase, and an iron sulfur oxidase (products of todA, todB, todC1, and todC2, genes, respectively).9 The toluene paramonooxygenase has been shown to be more complex, requiring the products of five genes (tmoABCDE) for activity. Both tod and tmo genes are chromosomally encoded. Several TOL-type plasmids have been described, such as XYL, pKJ1, pDK,

pWW53, pTK0, pDTG501, and pGB.12–20 Recently, a new plasmid TOM was reported that encodes a pathway that results in toluene hydroxylation at ortho and meta positions to yield 3-methylcatechol, which is then oxidized by TOM-encoded C23O to 2-hydroxy-6-oxohepta-2,4-dienoic acid.5,21 The genes that encode TOM and catechol 2,3dioxygenase are located on TOM (108 kb) of B. cepacia G4. The catabolic genes of ortho and meta cleavage of toluene have been worked out completely. In general, chromosomal genes encode the ortho pathway, while TOL plasmids encode the meta cleavage pathway. Benzoate, a product of toluene degradation, induces the expression of the genes of meta pathway and catechol, a product of toluene degradation, like benzoate, induces the ortho pathway (Figure 1). The genes encoding catabolic enzymes have been named the xyl genes and are organized into two operons referred to as the upper or the ortho and lower or meta pathways. The upper pathway genes xylCAB encode the degradation of toluene and xylene to benzoate and toluates, respectively. The lower pathway genes xylXYZLEGFJKIH encode the degradation of benzoate and toluates to acetaldehyde and pyruvate22 (Figure 1 and Table 1). The nucleotide sequence of the toluene/ benzene-2 monooxygenase locus has been determined in Pseudomonas sp. strain JS150.23 The degradation of benzene, toluene, ethylbenzene, and xylene in hypoxic environments has been studied by Kukor and Olsen,24 and they have deduced and compared the sequence of a catechol 2,3-dioxygenase functional in oxygen-limited environment with that of other extradiol oxygenases. Coschigano and Young25 studied the regulatory genes involved in the anaerobic toluene metabolism by a bacterium strain T1 by complementation experiments on the mutants of this strain. The monoxygenation by toluene and chlorobenzene dioxygenases during the metabolism of

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FIGURE 1. Pathway of toluene degradation in Pseudomonas putida mt-2. From Glazer et al. (1995). (Reproduced with permission from Microbial Biotechnology, Fundamentals of Applied Microbiology, p. 597. Copyright by W. H. Freeman and Company, New York, 1995.)

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TABLE 1 Enzymes and Regulatory Proteins Encoded by Genes on the TOL Plasmid pWW0 Gene

Enzyme or function

“Upper –pathway” operon

Enzymes involved in the conversion of toluene and xylenes to benzoate and toluates Xylene oxygenase Benzyl alcohol dehydrogenase Benzaldehyde dehydrogenase Enzymes involved in the degradation of benzoate and toluates to acetaldehyde and pyruvate Toluate di-oxygenase Catechol 2,3-dioxygenase 2-Hydroxymuconic semialdehyde dehydrogenase 2-Hydroxymunconic semialdehyde dehydrogenase 4-oxalocrotonate tautomerase 4-oxalocrotonate decarboxylase 2-Oxopent-4-enoate hydratase 2-Oxo-4-hydroxypentenoate aldolase Dihydroxycyclohexadiene carboxylate dehydrogenase Proteins involved in controlling the transcription of the upper- and lower–pathway genes Regulatory protein Regulatory protein

XylA XylB XylC “Lower (meta) – pathway” operon xylX,Y,Z XylE XylF XylG XylH XylI XylJ XylK XylL

XylR XylS

chlorotoluenes was reported by Lehning et al.26 The toluene/o-xylene monooxygenase purified from P. stutzeri OX1 displayed a very broad range of substrates and a peculiar regioselectivity.27 The studies have shown in some cases mechanisms by which bacteria degrade simple polycyclic aromatic compounds like naphthalene, phenanthrene, and anthracene have been subjects of interest for many years. Studies have revealed that there are at least two different sets of genes for the degradation of these compounds under aerobic conditions.28,29

B. Gene Regulation Regulatory elements of TOL plasmids have been named xylR and xylS.30 A model has been proposed for the regulation of the xyl genes (Figure 2). xylR is expressed constitutively at a high level in the presence of a substrate (e.g., toluene). When it enters, it

binds to the xylR protein to form a xyl-Rtoluene complex, which in turn binds to the promoter (Pupper) of the xylCAB operon and activates its transcription by influencing the binding of RNA polymerase. On the other hand, benzoate binds to the xylS protein and forms a xylS-benzoate complex, which binds to the promoter (Pmeta) of the lower pathway and activates the transcription of its genes. There are additional subtleties to the regulation. The xylR-toluene complex binds to the promoter (Ps) of the xylS operon as well as to the Pupper promoter. This activates the genes of both the upper and the lower pathways. The protein and the promoter sequence that regulate the xyl genes in P. putida appear to share a common evolutionary origin with the one used to regulate nitrogen metabolism in enteric bacteria, Klebsiella pneumoniae. In these bacteria, the transcription of genes like those for glutamine synthetase and nitrogenase is regulated by the availability of combined nitrogen. Two gene

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FIGURE 2. Model for xyl gene regulation in the TOL (p WW0) catabolic plasmid. Broken arrows indicate the direction of transcription. Solid arrows represent activation, and the open arrow represents the repression of the operon indicated. NtrA is a sigma factor produced constitutively by the host. In the presence of toluene, the XyIR-toluene complex activates transcription at the Pupper and P promoters. The consequent increased production of XylS leads to activation of transcription at Pmeta. Alternatively, benzoate in the presence of constitutive amounts of XylS is able to activate transcription at Pmeta XyIR functions as an autorepressor. (Based on Burlage, R. S., Hopper, P. W., and Sayler, G. S. (1989), Appl. Environ. Microbiol. 55, 1323–1328, Figure 5.)

products are required for the synthesis of N2-regulated proteins, namely, Ntr A (sigma factor) and Ntr B. Ntr A protein allows RNA polymerase to recognize a particular class of promoters. Only in the presence of a second protein does Ntr C RNA-polymerase-NtrA complex initiate transcription. In a similar fashion in P. putida, the maximal expression of the xylCAB and xylS operons requires NtrA sigma factor, and xylR acts as a NtrC activator protein. These two proteins are not required for transcription of the lower pathway genes (Figure 2). Wang et al.31 were the first to report the degradation of toluene and o, -m-, or p-xylenes by a marine oligobacterium Cycloclastricus oligotrophus RB1. They have reported five complete ORF’s designated as xylX, M, K, G, C1, and C2.

III. CHLOROBENZOATES These compounds are produced in vast amounts by the chemical industry for use as

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solvents, lubricants, plasticizers, and insulators as well as for use as herbicides. They are formed as key intermediates in the degradation of PCBs and benzoate herbicides. A number of reports have described isolation of 3-chlorobenzoate(3-CBA)-utilizing bacteria32–37 and 4-chlorobenzoate-(4-CBA) utilizing bacteria.38–43 The first report of an in vivo construction of a catabolic pathway for the mineralization of chlorinated aromatics, 4-CBA, is for Pseudomonas strain B13. This strain was originally collected by an enrichment culture technique with 3-CBA.33 It was found to oxidize 3-CBA to 3- and 4-chlorocatechol. However, it was unable to oxidize 4-chlorobenzoate or dichlorobenzoate (3,5-DCB). Because the benzoate 1,2-dioxygenase enzyme in this strain has a very narrow substrate specificity,44 it proved to be impossible to obtain either mutants or selectants of this strain that can degrade other chlorinated benzoates.45 The following pathway, the maleylacetate pathway, could metabolize

4-chloro or 3,5-dichlorocatechol, if they could be formed from 4-CB or 3,5-DCB.46–48 To circumvent the mentioned catabolic block, a 117-kb conjugative catabolic TOL plasmid pWWO was transferred into strain B13. This plasmid encodes complete utilization of toluene and m- and p-xylene, one step of which uses a nonspecific benzoate 1,2-dioxygenase.44 Transfer of the TOL plasmid into strain B13 and direct selection on 4-CBA resulted in a strain WR241.49 Further selection in the presence of 3,5-DCB led to the emergence of cells such as WR941, which could utilize this compound as the sole source of carbon and energy at a frequency of 10–8. A strain similar to WR941 has also been isolated from a mixed culture of soil organisms initially inoculated with both Pseudomonas sp. B13 and with P. putida PaWI, which was successively adapted to 3-CBA, 4-CBA, and 3,5-DCB in a chemostat.35 Chatterjee et al.50,51 followed the same procedure and isolated similar variants from their 3-CBA+ P. putida strain AC858, harboring the plasmid pAC25 (117 kb).32 Growth of AC858 in a chemostat in the presence of cells harboring the TOL plasmid allowed the emergence of cells that could also utilize 4-CBA in addition to 3-CBA.52 A. Organization and Regulation of Genes The organization and regulation of degradative genes for halogenated compounds have become clear from studies on the dissimilation of 3-CBA. Enzymes responsible for each step in the degradation of 3-CBA have been identified.53 Zaitsev and Baskunov54 reported a similar scheme for the metabolism of 3-CBA by Acinetobacter calcoaceticus. The genes involved in the degradation of 3-CBA have been shown to be plasmid encoded in the Pseudomonas sp. plasmids pAC25, pAC26, and pAC27.55,56 Operons for degradation pathways for 3-CBA

from P. putida, 1,2,4-TCB from a Pseudomonas sp., and benzoic acid from P. putida and Acinetobacter calcoaceticus are well characterized. In the above order, the related operons of the first two include clcABD, tcbCDEF,56–58 which convert 3-chlorocatechol, 3,4,6-trichloro-catechol, and 3,5-dichlorocatechol to maleylacetic acid, 5-chloromaleylacetic acid, and 2-chloromaleylacetic acid, respectively.46–48,57,58 The catechol degradative genes in the benzoate pathway of P. putida and A. calcoaceticus are clustered in dissimilar operons. In P. putida the catBC operons encode the conversion of the latter to beta-ketoadipate enol lactone, which is then transferred to beta-ketoadipate through the action of other genes on other loci.59 Catechol degradation in A. calcoaceticus is directed by the catA gene and the catBCEFD operons, which convert catechol, through beta-ketoadipate, to succinyl coenzyme A and acetyl coenzyme A.59 Transcription of the catBCEFD, catBC, and tcbCDEF operons is known to be controlled by the respective regulatory proteins CatM, CatR, and TcbR,60–62 each of which is a member of the LysR family of bacterial regulators.63 Coco et al.64 investigated the clcR gene that encodes LysR family activation of the clcABD chlorocatechol operon in Pseudomonas putida. Further studies by Coco et al.65 revealed that clcR exists as a dimer, like catR, but unlike catR binding of clcR to its target region (RBS an ABS) occurs in the absence of added effector. Recently, it has been shown that Pseudomonas putida P111 is able to utilize a broad range of monochlorinated, dichlorinated, and trichlorinated benzoates.43 That benzoate dioxygenase converts 3-CBA, 4-CBA, and benzoate to the corresponding catechols via reduction of a dihydrodiol was shown to be chromosomally coded and that chlorobenzoate-1,2-dioxygenase converts ortho-chlorobenzoates to the corresponding catechols was shown to be encoded on plasmid pPB111 (75 kb).

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The chlorobenzoate catabolic gene, cba, localized on a transposon Tn 5271 were subcloned on a broad host range expression vector in E. coli such that the new pathway extended to a range of chloroaromatics that were previously not degraded by the wild type.66 Expression studies of the cba C gene located on Tn 5271 revealed that the cba C dehydrogenase was required for growth on 3-CB but not 3,4-DCB.67 The broad specificity and high regiospecificity of orthohalobenzoate 1,2-dioxygenase was established in Pseudomonas aeruginosa that degrades 2,4-DCB and 2-CB.68 This feature of the enzyme indicated its importance for use in the degradation of mixtures of chlorobenzoates. As chlorobenzoates are substrates not easily metabolized in the environment by the existing bacteria, van der Meer69 postulated that the process of recombining or assembly of the existing genetic material must have occurred in many of them. This endows a bacterium to degrade simultaneously even unrelated compounds. Pseudomonas putida GJ31 was found to be able to grow on toluene and chlorobenzene. A study of the pathway of degradation in this bacterium revealed that the microbe had a meta-cleavage enzyme, resistant to inactivation by the acylchloride, thus providing the strain with the exceptional ability to degrade both toluene and chlorobenzene.70 This extradiol dioxygenase was purified and characterized to study its structural and kinetic properties.71 Burkholderia sp. strain P512 was also shown to attack a range of aromatic compounds, including chlorinated benzenes, toluene, biphenyls and dibenzo-p-dioxin, because of the presence of a broad spectrum chlorobenzene dioxygenase.72 Analysis of chlorobenzene-degrading transconjugants of Pseudomonas putida F1, which had acquired the genes for chlorocatechol degradation (clc) from Pseudomonas sp. strain B13 revealed that the clc gene cluster was present on a 105-kb amplifiable

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genetic element (named the clc element).73 The study suggested that the presence of multiple copies of the clc gene cluster was a prerequisite for the growth of P. putida RR22 on chlorobenzene and that amplification of the element was positively selected for in the presence of chlorobenzene. The degradation of 1,2,3,4-tetrachlorobenzene (TeCB) was studied in P. chlororaphis RW71.74 The compound, highly recalcitrant and hitherto not known to be degraded pollutant was mineralized by P. chlororaphis RW71. A functional chlorocatechol pathway was found to be induced in the microbe during growth on TeCB. Because complete metabolism of chlorinated benzenes is not a feature generally found in aerobic bacteria and is often thought to be due to novel recombination of two separate gene clusters,69 such a recombination could be responsible for adaptation of a natural microbial community in response to contamination with synthetic chemicals. The genetic information obtained for the chlorobenzoate degradation pathway in Ralstonia sp. JS705 revealed a unique combination of (partially duplicated) genes for chlorocatechol degradation and genes for a benzene-toluene type of aromatic ring dioxygenase.75 Such an evolution of the CB pathway seems to have created the capacity for natural attenuation of CBs at the contaminated sites.

IV. CHLOROPHENOXYACETATES Chlorinated derivatives of phenoxyacetates,such as 2,4-D and 2,4,5-T have been released into the environment as herbicides since 1950s. Unlike many of the recalcitrant synthetic compounds, 2,4-D is rapidly degraded by soil microorganisms.76,77 The degradation of 2,4,5-T, has been investigated to a lesser extent, and most of the information concerning the 2,4,5-T degradation has been

gathered by using reductive (anaerobic) sediments.78–80

A. Organization and Regulation of Genes Genes encoding 2,4-D degradation are present on conjugative plasmids.76,81–84 One such plasmid, pJP4 of Alcaligenes eutrophus,

has been studied extensively and has become a model for the study of 2,4-D degradation, which is encoded by tfd genes.85–89 It is an 80-kb, broad host range, PI incompatibility group plasmid. Restriction maps of this plasmid, and similar plasmids, have been published.85,86 It has been shown that pJP4 encodes enzymes for the conversion of 2,4-D to chloromaleylacetate (Figure 3). Enzymes responsible for further degradation via tri-

FIGURE 3. Restriction digest map of the plasmid pJP4. The positions of the fragments and the relative positions of the genes are from data reported elsewhere. The linear map represents the region encompassing the tfd genes. Intermediates produced and the respective genes and gene products responsible are shown on the right. (Filer and Harker, 1997, Appl. Env. Microbiol., 63, Figure 1, p. 318. Reproduced with permission from the American Society for Microbiology, USA.)

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carboxylic acid cycle intermediates are encoded by chromosomal genes of JMP134.86,90 The catabolic (tfd-ABCDEF) and regulatory (tfdR and tfdS) genes have been localized, sequenced, and characterized.78,85–88,91–97 All plasmids studied so far have been isolated from pure cultures that were obtained from environmental samples. More recently, Top et al.98 examined the diversity of 2,4-Ddegradative plasmids in the microbial community of an agricultural soil by complementation method using A. eutrophus as a recipient strain and they obtained transconjugates harboring the plasmids pTEM1 to pTEM7. It was found that unlike pJP4, pTEM1 did not appear to be an IncP1 plasmid and all the others from pTEM2 to pTEM7 to belong to the IncP1 group. They have concluded that the complementation method can be used to isolate other 2,4-D-degrading plasmids from soil microbial communities. Several other microorganisms, such as Acinetobacter, Arthrobacter, Corynebacterium, Flavobacterium, and Pseudomonas spp., have also been shown to degrade 2,4-D and related phenoxyacetates. Furthermore, the occurrence and nature of 2,4-Ddegradative plasmids in these microorganisms are not known. Chaudhry and Huang82 isolated a new 2,4-D-degradative plasmid, pRC10, from a Flavobacterium sp. This plasmid differs in size and restriction patterns from pJP4. It is a 45-kb plasmid, carries genes essential for the degradation of 3-CBA and 2-methyl-4-chlorophenoxyacetates (MCPA), imparts resistence to mercury, and encodes the utilization of 2,4-D. Comparison with plasmid pJP4 showed strong homology with the regions containing 2,4-Ddegradative genes, the first two genes responsible for the 2,4-D degradation, tfdA and tfdB. These have been cloned as a subfragment of the EcoRI A fragment of pRC10. Expression of pRC10 in P. putida (Nldr) and A. eutrophus JMP228 showed the cloned fragment coding for tfdA and tfdB.

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However, the expression of pRC10 in E. coli conferred on the cells only the resistence to mercury. The expression of tfd genes also has been found to be regulated by regulatory elements. It has been shown that tfdR regulates the expression of tfdA and the operon tfdCDEF but not of tfdB.94 The promoters for tfdA and the operon are homologous. Burkholderia cepacia AC1100 (formerly Pseudomonas cepacia AC1100) is known for its ability to utilize the recalcitrant 2,4,5-T as a sole source of carbon and energy.79 Genes involved in the conversion of 2,4,5-T to 2,4,5-trichlorophenol have been cloned, sequenced, and expressed.99,100 The tftA1 and tftA2 genes encode two subunits of the oxygenase enzyme responsible for this first step of the pathway,99 and their gene products showed homology to two multicomponent dioxygenases involved in benzoate and toluate degradation by Acinetobacter calcoaceticus (benAb) 101 and P. putida (xylXY).102 Recently, Daubaras et al.103 reported a unique gene cluster in B. cepacia AC1100 that is partially homologous to other degradative enzymes. The nucleotide gene sequence revealed the presence of six open reading frames designated ORF1 to ORF6. The two genes (ORF1 and ORF6) are homologous to genes in the tfdCDEF (tfdF and tfdC)96 and tcbCDEF (tcbF and tcbC)104 gene clusters; however, the organization of these are different. In tfd and tcb gene cluters, the catechol 1,2-dioxygenase genes (tfdC and tcbC) are found at the 5′ end of the cluster and the putative trans-chlorodienelactone isomerase genes (tfdF and tcbF) are found at the 3′ end of the cluster. In the 2,4,5-T gene cluster the genes are reversed, with the catechol 1,2-dioxygenase gene (ORF6) at the 3′ end of the cluster and the maleylacetate reductase gene (ORF1), which is homologus to the tfdF and tcbF genes, at the 5′ end of the cluster. Also, these genes in the 2,4,5-T gene cluster are separated by four ORFs, whereas in the other gene clusters there

are two or three ORFs in between. The other ORFs in the 2,4,5-T gene cluster encode unique enzymes compared with the cycloisomerase (TfdD and TcbD) and hydrolase (TfdE and TcbE) encoding genes of the tfd and tcb are homologous to the glutathione S-transferases, which have been identified in other degradative pathways. However, the genes encoding other glutathione S-transferase have not been associated with a cluster of genes or a gene encoding a glutathione reductase enzyme (ORF2); therefore, the 2,4,5-T gene cluster is a unique cluster of genes invloved in the degradation of a chlorinated aromatic compound.103

V. POLYCHLORINATED BIPHENYLS Polychlorinated biphenyl (PCBs) are a family of man-made compounds with excellent thermal stability and dielectric properties. Structurally, they are composed of biphenyl molecules carrying 1 to 10 chlorines. Depending on the number and position of the chlorines, 209 different PCBs congeners can be produced. According to one estimate, from 1929 to 1978, approximately 1.4 billion (635 million kg) of PCBs was produced, and it is estimated that several hundred million pounds have been released into the environment.

A. Organization of Catabolic Genes The vast majority of PCBs are in the form of commercial mixtures (such as arocolors) that contain 60 to 80 different PCB congeners and are thus particularly difficult to biodegrade.107 However, the cloning of genes responsible for the degradation of chlorobenzoate in organisms can lead to complete mineralization of PCBs. The involvement of plasmids in PCB degradation has been suggested for some bacteria such as

Klebsiella pneumoniae, Acinetobacter, and Alcaligenes sp., but the plasmids have not been characterized yet. Moreover, the enzymes and their corresponding genes have also not been isolated or characterized. Thus, the degradation of PCB is mediated by genes located primarily on the chromosome. Furukawa and Miyazaki106 have for the first time cloned and analyzed the expression of three genes (bphA, bphB and bphC) involved in biphenyl and PCB catabolism in P. pseudoalcaligenes KF707. Genes bphA, bphB, and bphC were found to be present on a 7.2-kb fragment in the order bphABC.107 The hydrolase activity, which converted the intermediate meta cleavage compounds to the final product, chlorobenzoic acid, was encoded by a putative bphD, which was missing from the cloned 7.2-kb fragment. Subsequently, the bphD gene was found not to be located downstream of bphC, but there was an extra DNA segment (bphX), approximately 3 kb, between bphC and bphD. The function of the putative bphX gene has not yet been elucidated. The bph operon in P. pseudoalcaligenes KF707 is thus organized as bphABCXD (Figure 4). The transcriptional initiation site is located 104 base pairs upstream from the start codon of the bphA cistron. Mondello108 has cloned the catabolic genes bphABCD encoding the entire pathway for the conversion of PCBs to chlorobenzoic acid in the Pseudomonas strain LB400. When Pseudomonas LB400 PCB-degrading genes were used as probes, significant hybridization of genomic DNA of A. eutrophus H80 occurred with them, indicating that PCBdegrading genes are similar in the two organisms.109 DNA from several other PCBdegrading strains showed no hybridization with this probe, which revealed the existence of distinct classes of genes encoding PCB degradation. It was further concluded that the biochemical similarities in PCB catabolism between LB400 and H80 reflect

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FIGURE 4. (a) Degradative pathway of biphenyl and PCB by soil bacteria and bph genes from P. pseudoalcaligenes. Top compounds. I. Biphenyl/PCB; II. 2,3-dihydroxy-4-phenylhexa-4,6-diene (dihydroxydiol compounds). III. 2,3-dihydrobiphenyl. IV. 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid. V. Chlorobenzoic acid. Enzymes: A, biphenyl dioxygenase; B, dihydrodiol dehydrogenase; C, 2,3-dihydroxybiphenyl dioxygenase; D, meta cleavage compound hydrolase, (b) Organization of the bph operon in P. pseudoalcaligenes KF707 and partial nucleotide sequence of the upstream region of the gene. +1, Transcription start site; RBS, putative ribosome binding site. (From Lal et al., 1995, Adv. Appl. Microbiol. 41, p. 80. Reproduced with permission from Academic Press, Inc., USA.)

their possession of closely related bph genes. Because it is extremely unlikely that these two strains have independently evolved such similar genes, the genes must have been acquired through some form of DNA transfer, suggesting their natural spread within bacterial populations in the environment. Hayase et al. 110 cloned the complete bphABCD genes from Pseudomonas putida KF715 by using bphACB and bphD of KF707

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as probes. This strain KF715 had the same gene order as strain LB400 in which bphABCD genes were cloned,108 but the restriction map of bphABCD of KF715 was quite different from that of LB400. However, like P. putida LB400, the extra DNA segment bphX observed in P. pseudoalcaligenes KF707 did not exist. The transcriptional initiation site of bphABCD (KF715) was identical with that of

bphABCXD in strain (KF707). Khan and Walia111 were able to clone bphABCD operon of P. putida OU83 in P. putida AC812, which was also expressed in E. coli. The unusual feature of this operon was the formation of 4-chloro-3-phenylcatechol, 4-chlorobenzoic acid, and benzoic acid from clones cultured in the presence of 4-chlorobenzophenyl. This indicated that the bph operon of P. putida OU83 has, in addition to bphABCD genes, a new dechlorinating gene that they named as dcpE. However, the precise order of genes has yet to be determined. bphABCD genes of P. putida OU83 have been used further to construct DNA probes for the detection and enumeration of PCBdegrading genotypes in microbial communities and for tracking GEM’s degrading PCB in the environment. As mentioned earlier, the reports on the degradation of PCB through genes that are located on the plasmids are few. Jones et al.112 reported a nontransmissible plasmid pWW1000 of 200 kb carrying genes required for biphenyl as well as 4-chlorobiphenyl catabolism in Pseudomonas sp. strain CB406. This plasmid was found to undergo deletions of its bph genes during growth on benzoate but was stable when grown on either succinate or nutrient-rich growth media. It is evident from the above discussion that the cloning of bph genes encoding enzymes that degrade PCB have considerable heterogeneity in their organization. One thing common in all the organisms so far studied is that bph genes are clustered and organized into single operons. However, restriction enzyme fragment profiles of the cloned DNAs specifying PCB degradation differ considerably in Pseudomonas sp. and Alcaligenes sp. Recently, Masai et al.113 cloned and sequenced the genes from a Gram-positive bacteria Rhodococcus sp. strain RHA1 isolated from a gamma-HCH contaminated site. They identified six bph genes, bphA1A2A3A4, bphB, and bphC that are re-

sponsible for the initial three steps of biphenyl degradation. The order of bph genes is bphA1A2A3A4-bphC-bphB and are different from that of other PCB degraders for which the order is bphA-bphB-bphC.114–117 The oxidation of PCBs was compared in Pseudomonas sp. strain 400 and P. pseudoalcaligenes KF707.118 The wide range of PCB congeners oxidized by LB400 and differences in substrate specificity was attributed to the differences in biphenyl 2,3-dioxyzenases of both organisms. On parallel grounds, the PCB-degrading strains were categorized into two groups based on the their ability to degrade 17 PCB congeners.119 Strains that degraded a broad range of PCBs but had relatively weak activity against di-, para-substituted PCBs were designated as having an LB400-type specificity. Strains designated as having a KF707-type specificity degraded a much narrower range of PCBs but had strong activity against certain di-, para-substituted congeners. The dehydrogenases involved in the bacterial degradation of aromatic compounds are related to each other and their phylogenetic relationships are very similar to the relationships observed for dioxygenases that catalyze the initial reaction in the degradation pathway. This was shown after a structural analysis of the 2,3-dihydro 2,3-dihydroxybiphenyl dehydrogenase of Comamonas testosteroni B356.120 Biphenyl degradation in Beijerinckia sp. strain B1 was studied by Kim and Zylstra.121 Initially, a small plasmid, pKG2, was implicated in the degradation of biphenyl by this strain. However, later studies revealed that at least two different operons on the chromosome were responsible for biphenyl degradation by Beijerinckia sp. strain B1. Most of the research on the biphenyl catabolic pathway was done on Gram-negative bacteria, especially the genus Pseudomonas. However, Masai et al.113 reported the degradation of polychlorinated biphenyl

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(including tri-, tetra-, and pentachlorobiphenyls), by a Gram-positive bacterium, Rhodococcus sp. strain RHA1. RHA1 exhibited high transformation activity on both ortho- and para-substituted PCB congeners. This was considered a superior characteristic of PCB degradation by RHA1 compared with other PCB degraders, including P. pseudoalcaligenes KF707 and Pseudomonas sp. strain LB400. The bph ABCD operon was cloned from RHA1 and expressed in E. coli, and it was found that the bph A and bph B played a more essential role in PCB degradation in RHA1. However, a more intensive study of the degradation pathway revealed that the bph genes in RHA1 were present on large linear plasmids.122 The Pulse Field Gel Electrophoresis (PFGE) of the RHA1 genome indicated that RHA1 had three large linear plasmids. bph ABC gene cluster was located on a 100-kb plasmid and bph DEF genes were borne by a 390-kb plasmid. Working on similar grounds, Kosono et al.123 reported plasmid borne degradation of biphenyl in Rhodococcus erythropolis TA421. These results confirmed that the biphenyl catabolic pathway in Rhodococcus strains are borne on plasmids. Rhodococcus sp. strain RHA1 was also found to harbor genes for the degradation of ethylbenzene (EB) in conjunction with biphenyl.124 The etb D1 and etb D2 genes were cloned from RHA1 and their nucleotide sequences determined. The etb genes were found to be present in the vicinity of the bph genes and exhibited similar induction patterns. Most of the highly chlorinated congeners, which can be degraded in liquid cultures, are not substantially degraded in soil, indicating that low bioavailability may have limited their degradation. Gilbert and Crowley125 showed that repeated application of carvone-induced bacteria enhanced the biodegradation of polychlorinated biphenyls in soil. This finding becomes all the more significant in light of the development of

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new technology that permits automated in situ fermentation and delivery of degrader microorganisms.

VI. NAPHTHALENE, PHENANTHRENE, AND ANTHRACENE Naphthalene is one of the aromatic hydrocarbons commonly identified in environmental samples and is identified as a priority pollutant in several countries.126 The genetics and biochemistry of bacteria capable of utilizing naphthalene as sole source for growth have been reviewed.127 Recently, the gene order of the nah operon has been reinvestigated by a combined genetic-biochemical approach.128

A. Organization of Genes Initially, strains of the bacteria Pseudomonas putida G7 (ATCC 17485) or pPG7 were isolated and this strain was found to metabolize naphthalene and salicylate.129 The degradation was plasmid mediated. This plasmid, which subsequently was studied in the derivatives of P. putida strain G1 (ATCC 17453), was named NAH7.130 In fact, the transfer of Nah+ Sal+ phenotype of PpG7 to derivatives of PpG7 and PpG1 by conjugation confirmed the role of this plasmid in the catabolism of naphthalene.131 Yen and Gunsalus132 mapped the genes encoding the enzyme for the first 11 steps of naphthalene oxidation in NAH7. Plasmid NAH7 is found to harbor genes of two operons.132 The upper pathway catabolic genes ABCDEF, which are responsible for the conversion of naphthalene to salicylate and the lower pathway catabolic genes GHIJK that convert salicylate to catechol.126 Catechol is then converted to intermediates of the Kreb’s cycle through meta pathway. All these genes are located on a 30-kb fragment of NAH7.

Other plasmids like NAH2 or pWW60, pDTG1, and pBS4 have been reported and characterized.133–136 In addition, a number of catabolic plasmids that degrade salicylate but not naphthalene have been reported. These plasmids have been primarily isolated from strains of P. putida.136,137 These plasmids are generally referred to as salycilate catabolic plasmids and have been discussed in detail by Yen and Serdar.127 Cane and Williams134 isolated mutants of pWW60-1 that after analysis revealed a deletion of 1.2 to 1.5 kb between nahG and the elimination of this fragment of DNA activated the genes of the meta pathway leading to a complete degradation of naphthalene. The gene order of the nah operon has been studied recently,128 and the proposed gene order for nah operon is nahABFCED (Figure 5). The restriction map of NAH7 plasmid also indicates that there are two regulatory genes nahN and nahL in the sal operon between the nahI and nahJ genes138 (Figure 6). DNA homology has also been found between NAH7 plasmid and other plasmids like pJP4, pAC27, and pWWO.78,91 Recently, the naphthalene dioxygenase genes (nahAa for ferredoxin NAP reductase and nahAB for ferre-

doxin NAP) from two strains of P. putida have been compared and show more than 90% homology.139 Several reports indicate the involvment of plasmids in the degradation of anthracene and phenanthrene.140–144

B. Regulatory Genes It was observed that salicylate, 2-aminobenzoic acid can activate the nah operons.145–147 Yen and Gunsalus148 reported that salicylate serves as an inducer for the sal operon enzymes, while naphthalene is not an inducer for enzymes of the nah operon. The regulatory gene nahR, which activates transcription of both the nah and sal operons, was cloned and mapped to determine the appropriate start site for transcription of the nah operon.138 The direction of the transcription of the nahR is opposite to that of the nahG.138 Because the amount of mRNA encoded by the nahR region is not significantly different in cells grown with and without salicylate, it appears that the nahR gene is transcribed constitutively and that the nahR protein can exist in two forms, an active form ( nahRa) and an inactive form (nahRi)

FIGURE 5. Naphthalene catabolic gene organization and regulation. (Southerland et al., 1995, Chap. 7, Figure 7.12, p. 294. Reproduced with permission from Wiley-Liss, Inc., Microbial Transformation and Degradation of Toxic Organic Chemicals, Young, L. Y. and Cerniglia, C. E., Eds.)

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FIGURE 6. Proposed metabolic pathway of aerobic degradation of gamma-HCH by Pseudomonas paucimobilis UT26. (From Nagata et al., (1994). J. Bacteriol. 176, 3117. Reproduced with permission from the American Society for Microbiology, USA.)

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that are in equilibrium.138 In the presence of a suitable inducer, nahRa predominates, and in its absence the equilibrium shifts toward nahRi.

VII. PARATHION Parathion is an organophosphorus insecticide that is extremely toxic to higher organisms because of its conversion to paraxon, a potent acetylcholinesterase inhibitor. Munnecke and Heish149 studied the degradation of parathion in a mixed microbial culture and found p-nitrophenol as the main metabolite. It has been observed that hydrolysis of parathion reduces its toxicity and releases p-nitrophenol and other watersoluble metabolites that can be further utilized by the microorganisms as a sole carbon and energy source. It was found that parathion hydrolase activity in Pseudomonas dimunita MG was plasmid encoded. This plasmid was designated as pCMSI and was found to be 70 kb in size.150 Subsequently, Mulbry et al.151,152 found that the Flavobacterium sp. ATCC 27551 contains a 39-kb plasmid pPDL2 that is involved in the degradation of parathion. The gene encoding parathion hydrolase from pCMSI was termed opd (organophosphorus-degrading gene), and was shown to have homology with pPDL2.151 Further, DNA hybridization studies have revealed that these plasmids share homology only in an approximately 5.1-kb region.152 The hydrolase-producing Flavobacterium strain has been used in a pilot-scale system to detoxify waste containing organophosphate insecticide caumaphos.153 However, no further work was done in order to utilize this strain for decontamination of parathion under normal conditions.

VIII. HEXACHLOROCYCLOHEXANE HCH is still in use in the tropical and subtropical countries like India. Anaerobic

degradation of HCH isomers has been studied extensively. However, only a few reports of aerobic degradation of HCH isomers are available.154–159 The isolation of microbes for the aerobic degradation of gammaHCH made it possible to design the experiments for cloning of catabolic genes responsible for the degradation of this compound. Imai et al.156 initially characterized the degradative pathway of lindane in Pseudomonas UT26. A genomic library of Pseudomonas paucimobilis UT26 was constructed for P. putida by using the broad-host-range cosmid vector pKS13. One of the clones was further characterized for subcloning the lindane-degradative gene. This clone was shown to contain a recombinant plasmid (pKSR1) that was actually pKS13 with a 25-kb insert. From pKSR1, a 5-kb Hind III fragment was subcloned into pUC118 (pIMAI). E. coli cells containing pIMAI retained the ability to convert lindane to 1,2,4-trichlorobenzene (1,2,4-TCB). This result showed that 5-kb Hind III fragment was reponsible for the transformation of lindane to 1,2,4-TCB. A series of deletions were introduced in the 5-kb insert of pIMAI by nuclease digestions. It was concluded, that a 500-kb fragment contained the region for the activity that converted lindane to 1,2,4-TCB. The nucleotide sequence of this region and its flanking regions was determined. Only one open reading frame of 465 bp was found within the 500-bp region that encodes for gamma-HCH dehydrochlorinase, which confers deydrochlorinaton yielding 1,2,4-TCB from lindane.156 In Pseudomonas paucimobilis UT26 lindane is converted by two steps of dehydrochlorination to a chemically unstable intermediate, 1,3,4,6-tetrachloro-1,4-cyclohexadiene (1,4-TCDN), which is then metabolized to 2,5-dichloro-2,5-cyclohexadiene1,4-diol (2,5-DDOL) by two steps of hydrolytic dehalogenation via the chemically unstable intermediate 2,4,5-trichloro-2,5cyclo-hexadiene-1-ol (2,4,5-DNOL). To clone a gene encoding the enzyme respon261

sible for the conversion of 1,4-TCDN and 2,4,5-DNOL, a genomic library of P. paucimobilis UT 26 was constructed in P. putida PpY101 LA, into which a gene had been introduced by Tn5. An 8-kb BglII fragment from one of the cosmid clones, which could convert lindane to 2,5,-DDOL, was subcloned.157 Subsequent deletion analysis revealed that a 1.1-kb region was responsible for the activity. Nucleotide sequence analysis revealed an open reading frame (designated the lin b gene) of 885 bp within the region. The deduced amino acid sequence of lin B showed significant similarity to hydrolytic dehalogenase, DhlA.160 The protein product of the lin b gene is a 32-kDa protein, as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Not only 1-chlorobutane, but also 1-chlorodecane (C10) and 2-chlorobutane, which are poor substrates for other dehalogenases, were good substrates for lin B, suggesting that lin B may be a member of haloalkane dehalogenases with broad-range specificity for substrates.157 Further, Nagata et al.161 cloned lin c gene encoding 2,5-DDOL dehydrogenase, which converts 2,5-DDOL to 2,5-dichlorohydroquinone (2,5-DCHQ) in P. paucimobilis. Recently, Miyauchi et al.162 cloned and characterized lin D gene that encode the conversion of 2,5-DCHQ rapidly to chlorohydroquinone (CHQ) and also converts CHQ slowly to hydroquinone. It was shown that Lin D activity in crude cell extracts was increased 3- to 7-fold by the addition of glutathione. The pathway of gamma-HCH degradation was reconstructed, as shown in (Figure 6).

IX. RECENT DEVELOPMENTS, CONCLUSIONS, AND FUTURE PROSPECTS Biodegradation and its application in bioremediation hold promise for efficient and

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cost-effective means for cleaning contaminated environments. Biosensors to detect catabolic genes have been prepared for naphthalene, salicylate, xylene, and toluene.163–166 Burlage et al.167 demonstrated subcloning of the promoter from the upper pathway of NAH7 to form a fusion with the luxCABE genes of Vibrio fischeri. A fragment from NAH7, which contains the promoter for the upper pathway of degradation, was transcriptionally fused to the lux genes of V. fischeri. A Pseudomonas strain containing this construct in plasmid pUTK9 was inducible to high levels of production in the presence of a suitable substrate and the nahR regulatory gene product. Induction of bioluminescence was found to coincide with naphthalene degradation. Naphthalene metabolism was greatest when the growth rate was slow, such as during stationary phase or in nutrient poor medium. Subsequently, King et al.165 described the construction and characterization of bioluminescent reporter plasmid pUTK21 for naphthalene degradation. This plasmid was developed by transposon (Tn4431) insertion of the lux gene cassette from V. fischeri into a naphthalene catabolic plasmid in P. fluorescence. The insertion site of the lux transposon was the nahG gene encoding for salicylate hydroxylase. Luciferase-mediated light production from P. fluorescence strains harboring this plasmid was induced on exposure to naphthalene or the regulatory inducer metabolite, salicylate. In continuous culture, light induction was rapid and was highly responsive to dynamic changes in naphthalene exposure. Strains harboring pUTK21 were responsive to aromatic hydrocarbon contamination in soils from manufactured gas plants and produced sufficient light to serve as biosensors of naphthalene exposure and reporters of naphthalene biodegradative activity. The use of bioluminescent light as a measure of catabolic activity offers attractive applications in environ-

mental simulations and potential field analysis of microbial degradative activity. In comparison to conventional activity assays, bioluminescent reporter systems are also noninvasive, nondestructive, rapid, and population specific. An attempt has been made to restrict horizontal gene spread in the environment and to create organisms whose behavior in the field is more predictable. Munthali et al.168 have designed a gene containment system. Use of this system is exemplified by the construction of microorganisms designed to degrade PCB. To reduce such gene transfer from bacteria introduced in the environment, a gene containment system based on posttranslation inhibition of a universal lethal function (colicin E3 RNase) by a cognate immunity function (immunity E3 protein) was designed and demonstrated to inhibit severe plasmid transfer in vitro.169 Further, Munthali et al.168,170 combined a mini-Tn5 cloning system that allows stable insertion of foreign genes into the chromosomes of a variety of Gram-negative bacteria. The molecular studies on the various processes and products of gene expression in the enviornment would yield information that can assist in understanding, and consequently manipulating, remediation in situ. At the gene level, detection of biodegradation genes in indigenous and introduced microbes reveals the diversity of degrading populations and pathways, identifies strains with novel catabolic pathways, and follows the establishment of introduced and indigenous biodegrading species in treatment sites. Molecular tools like biosensors assist in identifying conditions conducive for induction of catabolic pathways and confirm that biodegradative genes are indeed transcribed in situ. It is evident from the literature presented herein that the characterization and regulation of genes for some of the priority pollutants like naphthalene, chlorobenzoates, PCBs, and chlorophenoxyacetates have been partially com-

pleted in the last 2 decades. Such a study of catabolic genes and enzymes has provided the basic information for the understanding of the genetic aspects of degradation. However, less has been done on the catabolic genes of HCH and no information is available for DDT.171 In future work, more emphasis should be given in this direction, as organochlorines are still widespread in the subtropical and Third World countries. Microorganisms are exposed to an immense variety of organic compounds in the environment. Rigorous assessment of the physical and biological factors that contribute to the disappearance of xenobiotics in the environment is challenging. Genetic manipulation offers a way of engineering microorganisms to deal with a pollutant that may be present in the waste stream from an industrial process. The simplest approach is to extend the degradative capabilities of existing metabolic pathways within an organism either by introducing additional enzyme from other organisms or by modifying the specificity or catalytic mechanisms of enzymes already present. The continuously expanding knowledge on catabolic pathways and critical enzymes is providing the basis for the rational genetic design of new and improved enzymes and pathways for the development of more performant processes. There have been some intitial successes in bioremediation over the last few years, and as a result new bioremediation companies have started up. Despite the obvious advantage of bioremediation and its success, however, most companies experience problems regarding the recongnition of the utility and the acceptability of bioremediation technology.

ACKNOWLEDGMENTS Atul Johri gratefully acknowledges UNESCO for providing the short-term Re-

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search Fellowship in Biotechnology at the Department of Chemical Engineering, University of Waterloo,Waterloo, Ontario, Canada. Meenakshi Dua acknowledges the GATE-UGC, Govt. of India, for providing the research fellowship.

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